BCMA-targeted bortezomib nanotherapy improves therapeutic efficacy, overcomes resistance, and modulates the immune microenvironment in multiple myeloma

Bortezomib (BTZ) is a standard-of-care treatment in multiple myeloma (MM); however, adverse side effects and development of resistance limit its long term benefit. To improve target specificity, therapeutic efficacy, and overcome resistance, we designed nanoparticles that encapsulate BTZ and are surface-functionalized with BCMA antibodies (BCMA-BTZ-NPs). We confirmed efficient cellular internalization of the BCMA-BTZ-NPs only in BCMA-expressing MM cells, but not in BCMA-knockout (KO) cells. In addition, BCMA-BTZ-NPs showed target-specific cytotoxicity against MM cell lines and primary tumor cells from MM patients. The BCMA-BTZ-NPs entered the cell through receptor-mediated uptake, which escapes a mechanism of BTZ resistance based on upregulating P-glycoprotein. Furthermore, BCMA-BTZ-NPs induced cell death more efficiently than non-targeted nanoparticles or free BTZ, triggering potent mitochondrial depolarization followed by apoptosis. In BTZ-resistant cells, BCMA-BTZ-NPs inhibited proteasome activity more effectively than free BTZ or non-targeted nanoparticles. Additionally, BCMA-BTZ-NPs enhanced immunogenic cell death and activated the autophagic pathway more than free BTZ. Finally, we found that BCMA-BTZ-NPs selectively accumulated at the tumor site in a murine xenograft model, enhanced tumor reduction, and prolonged host survival. These results suggest BCMA-BTZ-NPs provide a promising therapeutic strategy for enhancing the efficacy of BTZ and establish a framework for their evaluation in a clinical setting.


Cells and Materials
MM.1S, H929, RPMI 8226, and human dermal fibroblasts (HDF) cells were procured from the American Type Culture Collection (ATCC).AMO-1 was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ).All cell lines were first tested for mycoplasma using the MycoAlert Mycoplasma Detection Kit (Lonza) and further validated by short tandem repeat (STR) DNA fingerprinting analysis (Molecular Diagnostic Laboratory, DFCI).All the cells except HDF cells were cultured in Dulbecco's modified Eagle's medium (DMEM) and were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 1× antibioticantimycotic, 1× GlutaMAX, and 1× Hepes at 37°C in 5% CO2.
All chemicals were purchased from Sigma (St. Louis, MO, USA), and antibodies were procured from Biolegend unless otherwise mentioned.Alexa Fluor 488 and Alexa Fluor 647 were purchased from Life Technologies (Carlsbad, CA, USA).The CellTiter-Glo®2.0 assay was obtained from Promega (Madison, USA).Caspase 3, 8, and 9 colorimetric assay kit was obtained from Abcam (USA).

Synthesis and Physical characterization of BCMA-conjugated nanoparticles
Primary emulsion was developed by dropwise addition of PBS in DCM organic phase containing PLGA and BTZ under sonication at 100W for 90-180s.The primary emulsion (w/o) was then dropwise added to 4ml of aqueous phase containing 2.5% PVA (w/v) under the same sonication condition.The BCMA antibody was conjugated on the surface of the nanoparticles using the EDC/NHS coupling reaction.
The particle size and surface charge were measured using the dynamic light scattering instrument Zetasizer Nano-ZS90 (Malvern Instruments; Westborough, MA).To determine the surface morphology and nanoparticle structure, the lyophilized powder was scanned under Transmission electron microscopy (TEM) (JEM-1000, JEOL, Tokyo, Japan).We optimized the initial drug feed to confirm the highest drug loading in the nanoformulation.Bortezomib loading in the nanoparticles was analyzed at absorbance maxima (λ = 270 nm) with an Infinite 200 Pro plate reader (Tecan).The NIR dye, DiR (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindotricarbocyanine Iodide)loaded nanoparticles were prepared using a similar method of drug-loaded nanoparticle preparation.
The in vitro drug release kinetics of BTZ-nanoparticles were determined at different timepoints by collecting the supernatant from BTZ nanoparticles dissolved in PBS and incubated at 37°C under shaking to determine the drug concentration in the supernatant at λ = 270 nm using a plate reader.Antibody conjugation onto the nanoparticle surface was analyzed by orbitrap mass spectrometers (Thermo Scientific) at the Taplin Biological Mass Spectrometry Facility, Harvard Medical School.Antibody-conjugated nanoparticles and unconjugated particles were analyzed by western blotting.
Additionally, we determined the covalently conjugated surface monoclonal antibody concentration of the nanoparticles by protein assay.

CellTiter-Glo cell viability assay
Cells were seeded in their respective medium (10000 cells per well, in a 100 µl final volume) in 96-well corning plates followed by treatment with free drug, different types of nanoformulations, and with verapamil.In the case of co-culturing MM cells and BMSCs, BMSCs were seeded in a 96-well corning plate, and 24h later, MM cells and different treatment groups such as BTZ, BTZ-NP, and BCMA-BTZ-NP were added followed by co-incubation for 24h.After treatment, plates were equilibrated to room temperature after the 37°C incubator, and 100 µl of CellTiter-Glo reagent (Promega) was added to each well.Plates were gently shaken on an orbital shaker for 1 min at 500 rpm and then incubated at room temperature for 10 min.To determine the effect of soluble BCMA on cytotoxicity of BTZ, BTZ-NP, and BCMA-BTZ-NP, recombinant BCMA (Peprotech) was added to the culture media in a concentration range from 0-1000 ng/ml and 0-25 µg/ml.Luminescence was determined by a spectrophotometer (SpectraMax M3, Molecular Devices).

FACs and Confocal Microscopy-Based Assay
Cells were collected (15,000-20,000) after treatment with dye-conjugated nanoparticles and cytospun for 5 minutes at 800 rpm, followed by fixation with 4% paraformaldehyde at room temperature for 15 minutes and washing three times with 1% FBS in PBS.For detection of acidic vesicular organelles, acridine orange staining was performed following the manufacturer's protocol (Thermo Fisher Scientific).Autophagic flux was monitored by staining autophagosome and autolysosome compartments with the Cyto-ID Green fluorescent probe (Enzo Life Sciences, Farmingdale, NY), as per manufacturer's protocol.For endosome staining or PgP staining, specific dyeconjugated antibodies were used after fixation.Nuclei were co-stained with Fluoro-gel II mounting medium with DAPI (Thermo Fisher Scientific), and images were captured using the Yokogawa Spinning Disk Confocal/TIRF System and analyzed with GIMP software.

Proteasomal Activity Assay
Bortezomib-sensitive and -resistant cells were treated with free drug bortezomib BTZ-BTZ-NPs, and BCMA-BTZ-NPs, and then analyzed for chymotrypsin-like proteasome activity using the luminogenic proteasome substrate-based Proteasome-Glo Chymotrypsin-Like Cell-Based Assay Kit (Promega), as per the manufacturer's instructions.Luminescence was quantified using a 96-well plate reader.

ROS generation
Apoptosis induction after treatment was evaluated by Annexin-V/PI staining and flow cytometric analysis, as previously described.The cationic lipophilic dye 5,5′,6,6′tetrachloro-1,1′,3,3′-tetraethyl benzimidazolylcarbocyanine iodide (JC-1, Sigma-Aldrich) was used to monitor the mitochondrial transmembrane potential (ΔΨm) following the previously mentioned procedure.ROS generation was assessed in BTZsensitive and -resistant cells, after incubation with BTZ, BTZ-nanoparticles, and verapamil, alone or in combination, by monitoring the hydrogen peroxide concentration using the ROS-Glo™ H2O2 Assay (G8820, Promega) kit following the manufacturer's protocol.Luminescence was determined using a plate reader.

Measurement of Caspases activation
Cell lysates were prepared from MM.1S and H929 cells after treatment with free drug, BTZ, BTZ-NPs, and BCMA-BTZ-NPs using RIPA buffer, and protein concentration was determined.Each sample with 150 µg of protein was aliquoted, and DTT with respective DEVD-p-NA substrate were added according to the manufacturer's protocol.
Samples were mixed well and incubated at 37°C for 90 min.The OD was measured at